We propose to develop a new experimental platform for the study of biofilms. Biofilms consist of bacterial consortia living on surfaces. Their inherent tolerance to host defenses and increasing resistance to antibiotics cause growing concern in many clinical applications, including pathogenesis of infectious diseases and clinical infection of prosthetic implants and biomaterials. To date, the small spatial scales, heterogeneity and time-dependence have defeated the rationalization of biofilm processes in terms of general mechanistic principles, forcing therapeutics to resort primarily to empirical strategies of limited success. Current experimental techniques provide only crude means of controlling a biofilm's microenvironment and are severely limited in quantifying microscale processes at the single-cell level with appropriate spatiotemporal resolution. Our approach is to integrate two state-of-the-art experimental techniques, microfluidics and digital holographic microscopy, to create a powerful new platform for biofilm studies. We call this 5Fluidic-DHM. 5Fluidic-DHM will exploit the versatility of microfluidics in accurately manipulating microenvironmental conditions, including geometrical, chemical and fluid dynamical parameters, coupled with the ability of digital holography to capture three-dimensional dynamics at single-cell level and high temporal resolution. Our goal for this R21 project is to develop, validate and optimize a 5Fluidic-DHM platform. We will showcase the advantages of this approach by testing it on two important biofilm processes: cell attachment to surfaces and flow through biofilm water channels. This project is directly relevant to human health, as it will improve our ability to treat biofilm- originated infection by advancing the state-of-the-art in biofilm experimentation with an instrument of unprecedented accuracy, resolution and flexibility. This will ultimately lead to enhanced therapeutic strategies in a wide range of clinical applications.